Acrolein is a highly reactive α,β-unsaturated aldehyde and is known to react with DNA forming exocyclic acrolein-deoxyguanosine adducts (Acro-dG). These aldehyde-DNA lesions may play a role in mutagenesis, carcinogenesis, and neurodegenerative diseases. In the present work, we described the development and evaluation of a highly sensitive and selective capillary liquid chromatography nanoelectrospray isotope dilution tandem mass spectrometry method for quantitatively analyzing Acro-dG in DNA hydrolysates. This was achieved by applying a stable isotope-labeled analogue Acro-dG- 13C10, 15N5 as an internal standard to the DNA to be analyzed and then hydrolyzing the DNA enzymatically to nucleosides. The acrolein-modified nucleosides were separated from normal nucleosides by capillary liquid chromatography and quantified by a high-capacity ion trap mass spectrometer in the MS/MS mode. The developed method achieved attomole-level sensitivity (limit of detection was 10 fg, 31 amol on column) for detection of pure Acro-dG adduct standards. The limit of quantification of Acro-dG adducts obtained in 10 μg of DNA hydrolysates was 1.5 fmol, which corresponded to 50 adducts/109 normal nucleosides. Application of this method to the analysis of Acro-dG adducts in acrolein (10-fold)-treated calf thymus DNA showed ∼830 lesion/106 DNA nucleosides using as low as 50 ng of DNA. Application of this method to DNA samples (1-2 μg) isolated from brain tissues from Alzheimer's disease subjects and age-matched controls demonstrated 2800-5100 Acro-dG adducts/109 DNA nucleosides. Statistically significant differences (P < 0.05) in levels of Acro-dG between AD subjects and controls were observed in DNA isolated from the hippocampus/ parahippocampal gyrus.
|Number of pages||8|
|State||Published - Sep 15 2005|
ASJC Scopus subject areas
- Analytical Chemistry